Current 2D co-culture models for examining osteocyte-osteoblast interactions and their response to environ- mental stimuli cannot recapitulate the 3D cellular syncytium formed by these cells in the skeleton, representing a fundamental gap in our approach for elucidating regulatory mechanisms of in vivo bone formation. Because bone cells behave differently in 2D than in 3D conditions, continued existence of this gap represents a significant barrier that will continue to limit development of therapies harnessing anabolic pathways to treat and pre- vent skeletal wasting diseases, like osteoporosis. The long-term research goal motivating the studies in this proposal is to develop new in vivo physical and pharmacological therapies to treat skeletal disease through an understanding of osteocyte regulation of bone-forming osteoblasts. The objective of this proposal is to develop a novel 3D co-culture system that reproduces in vivo osteocyte-osteoblast interactions and their responses to osteocyte-directed physical stimuli. The central hypothesis is that this 3D co-culture system will reproduce in vivo osteocyte physiology and gap junction-mediated regulation of osteoblasts in response to osteocyte- directed fluid flow. The rationale for these studies is that this innovative 3D co-culture system will transform how physical and biochemical interactions among bone cells can be examined in a realistic in vitro environment. Guided by the research team's experience in skeletal mechanobiology, in vitro fluid loading models, and computational mechanical analyses, the central hypothesis will be tested by pursuing three specific aims: (1) Develop a 3D composite construct that models native bone architecture and reproduces in vivo osteocyte physiology; (2) Characterize the autocrine response of matrix-embedded osteocytes to fluid flow through the 3D construct; and (3) Characterize the gap junction-mediated paracrine response of osteoblasts to osteocyte- directed fluid flow in 3D. In Aim #1, the composition of the 3D mineral-collagen construct will be optimized to promote bone formation and osteocyte differentiation. In Aim #2, the autocrine response of matrix-embedded osteocytes to lacunar canalicular fluid shear stress will be determined by measuring real-time changes in canonical Wnt-catenin signaling through Sost and TCF/Lef activity. In Aim #3, osteoblasts will be seeded on the osteocyte-enriched 3D construct, but shielded from direct fluid flow, to examine the gap junction-mediated response of osteoblasts to osteocyte-directed flow stimuli. The research proposed in this application is innovative because it will validate a novel 3D mineral-collagen co-culture system to elucidate the role of osteocyte- directed physical stimuli in regulating bone formation and is unique among 3D bone tissue co-culture models in its flexibility to incorporate bone cells from various sources and genetic models. The proposed research is significant because incorporating primary human or transgenic mouse cells in future applications of this novel technology will accelerate translational research aimed at discovering the mechanistic role of cellular transcription factors and evaluating their efficacy as targets for pharmacologic therapies for skeletal wasting diseases.